The aim of our research program is to gain a clearer understanding of the mechanisms of regulation of cholesterol biosynthesis. We will focus on regulation of transcription of the gene for the rate limiting enzyme of cholesterol biosynthesis, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase). We will also determine how HMG-CoA reductase is inserted into the endoplasmic reticulum and how cholesterol interacts with this membrane protein to alter its degradation rate. We have isolated a cell line, ML100, which is resistant to compactin and overproduces HMG-CoA reductase due to an increased rate of transcription. We will determine why the transcription rate for the gene is elevated in this cell line. We will localize the "signal" domain of HMG-CoA reductase which is necessary for insertion of the polypeptide into the endoplasmic reticulum. This will be done by in vitro mutagenesis of the cDNA clone for HMG-CoA reductase followed by expression in animal cells. In addition, the portion of the wild type or mutagenized gene for the membrane domains will be fused to Beta-galactosidase and this system used to determine localization of the polypeptide. We will use in vitro mutagenesis and expression experiments to localize the region within the membrane domain of the HMG-CoA reductase polypeptide responsible for cholesterol mediated regulation of degradation. We will synthesize fluorescent derivatives of compactin to use in a cell sorter method of detecting novel mutatn cell lines altered in the regulation of the levels of HMG-CoA reductase.